Consumable for laser capture microdissection

ABSTRACT

Systems and methods for acquiring laser capture microdissection samples are disclosed. A scattering media is located within a beam path defined by the laser capture microdissection microscope and the sample is imaged through the scattering media. A transfer film carrier includes a substrate surface and a laser capture microdissection transfer film coupled to the substrate surface of the transfer film carrier. The scattering media is optically coupled to a laser capture microdissection transfer film being located in or attached to the laser capture microdissection transfer film or transfer film carrier. The systems and methods provide improved sample imaging and facilitate quick and accurate laser capture microdissection while simultaneously minimizing contamination.

CROSS-REFERENCE TO RELATED APPLICATION

This application is a divisional of copending U.S. patent applicationSer. No. 08/984,979 filed on Dec. 4, 1997 which is acontinuation-in-part U.S. Provisional Patent Application Ser. No.60/060,732, filed Oct. 1, 1997, the entire contents of both of which arehereby incorporated by reference as if fully set forth herein.

BACKGROUND OF THE INVENTION

1. Field of the Invention

The present invention relates generally to the field of laser capturemicrodissection (LCM). More particularly, the present invention relatesto apparatus for acquiring LCM samples that include an LCM film mountedon at least a part of the interior of an analysis container.Specifically, a preferred implementation of the present inventionrelates to a substantially planarized ethylene vinyl acetate (EVA)polymer LCM film that is hot vacuum baked onto the bottom of amicrocentrifuge tube cap. The present invention thus relates to an LCMsample acquisition apparatus of the type that can be termed planar cap.

2. Discussion of the Related Art

Diseases such as cancer have long been identified by examining tissuebiopsies to identify unusual cells. The problem has been that there hasbeen no satisfactory prior-art method to extract the cells of interestfrom the surrounding tissue. Currently, investigators must attempt tomanually extract, or microdissect, cells of interest either byattempting to mechanically isolate them with a manual tool or through aconvoluted process of isolating and culturing the cells. Mostinvestigators consider both approaches to be tedious, time consuming,and inefficient.

A new technique has been developed which can extract a small cluster ofcells from a tissue sample in a matter of seconds. The technique iscalled laser capture microdissection (LCM). Laser capturemicrodissection is a one-step technique which integrates a standardlaboratory microscope with a low-energy laser and a transparent ethylenevinyl acetate polymer thermoplastic film such as is used for the plasticseal in food product packaging.

In laser capture microdissection, the operator looks through amicroscope at a tissue biopsy section mounted on a standard glasshistopathology slide, which typically contains groups of different typesof cells. A thermoplastic film is placed over and in contact with thetissue biopsy section. Upon identifying a group of cells of interestwithin the tissue section, the operator centers them in a target area ofthe microscope field and then generates a pulse from a laser such as acarbon dioxide laser having an intensity of about 50 milliwatts (mW) anda pulse duration of between about 50 to about 500 milliseconds (mS). Thelaser pulse causes localized heating of the plastic film as it passesthrough it imparting to it an adhesive property. The cells then stick tothe localized adhesive area of the plastic tape directly above them,whereupon the cells are immediately extracted and ready for analysis.Because of the small diameter of the laser beam, extremely small cellclusters may be microdissected from a tissue section.

By taking only these target cells directly from the tissue sample,scientists can immediately analyze the gene and enzyme activity of thetarget cells using other research tools. Such procedures as polymerasechain reaction amplification of DNA and RNA, and enzyme recovery fromthe tissue sample have been demonstrated. No limitations have beenreported in the ability to amplify DNA or RNA from tumor cells extractedwith laser capture microdissection.

Laser capture microdissection has successfully extracted cells in alltissues in which it has been tested. These include kidney glomeruli, insitu breast carcinoma, atypical ductal hyperplasia of the breast,prostatic interepithielial neoplasia, and lymphoid follicles. The directaccess to cells provided by laser capture microdissection will likelylead to a revolution in the understanding of the molecular basis ofcancer and other diseases, helping to lay the groundwork for earlier andmore precise disease detection.

Another likely role for the technique is in recording the patterns ofgene expression in various cell types, an emerging issue in medicalresearch. For instance, the National Cancer Institute's Cancer GenomeAnatomy Project (CGAP) is attempting to define the patterns of geneexpression in normal, precancerous, and malignant cells. In projectssuch as CGAP, laser capture microdissection is a valuable tool forprocuring pure cell samples from tissue samples.

The LCM technique is generally described in the recently publishedarticle: Laser Capture Microdissection, Science Volume 274, Number 5289,Issue 8, pp 998-1001, published in 1996, the entire contents of whichare incorporated herein by reference. The purpose of the LCM techniqueis to provide a simple method for the procurement of selected humancells from a heterogeneous population contained on a typicalhistopathology biopsy slide.

A typical tissue biopsy sample consists of a 5 to 10 micron slice oftissue that is placed on a glass microscope slide using techniques wellknown in the field of pathology. This tissue slice is a cross section ofthe body organ that is being studied. The tissue consists of a varietyof different types of cells. Often a pathologist desires to remove onlya small portion of the tissue for further analysis.

LCM employs a thermoplastic transfer film that is placed on top of thetissue sample. This film is manufactured containing organic dyes thatare chosen to selectively absorb in the near infrared region of thespectrum overlapping the emission region of common AlGaAs laser diodes.When the film is exposed to the focused laser beam the exposed region isheated by the laser and melts, adhering to the tissue in the region thatwas exposed. The film is then lifted from the tissue and the selectedportion of the tissue is removed with the film.

Thermoplastic transfer films such as a 100 micron thick ethyl vinylacetate (EVA) film available from Electroseal Corporation of PomptonLakes, N.J. (type E540) have been used in LCM applications. The film ischosen to have a low melting point of about 90° C.

The thermoplastic EVA films used in LCM techniques have been doped withdyes, such as an infrared napthalocyanine dye, available from AldrichChemical Company (dye number 43296-2 or 39317-7). These dyes have astrong absorption in the 800 nm region, a wavelength region thatoverlaps with laser emitters used to selectively melt the film. The dyeis mixed with the melted bulk plastic at an elevated temperature. Thedyed plastic is then manufactured into a film using standard filmmanufacturing techniques. The dye concentration in the plastic is about0.001 M.

While the films employed in LCM applications have proved satisfactoryfor the task, they have several drawbacks. The optical absorption of adye impregnated film is a function of its thickness. This property ofthe film may be in conflict with a desire to select film thickness forother reasons.

The organic dyes which are used to alter the absorption characteristicsof the films may have detrimental photochemistry effects in some cases.This could result in contamination of LCM samples. In addition, theorganic dyes employed to date are sensitive to the wavelength of theincident laser light and thus the film must be matched to the laseremployed.

BRIEF SUMMARY OF THE INVENTION

An object of the invention is to improve the speed of the laser capturemicrodissection technique. Another object of the invention is to improvethe accuracy of the laser capture microdissection technique. Anotherobject of the invention is to improve the reproducibility of the lasercapture microdissection technique. Yet another object of the inventionis to reduce the amount of contamination involved with the laser capturemicrodissection technique. Another object is to improve the imaging ofthe sample. Therefore, there is a particular need for an LCM consumablethat integrates an LCM film into the interior of an analysis container.A planar cap includes a substantially planarized ethylene vinyl acetate(EVA) polymer LCM film that is hot vacuum baked onto the bottom of amicrocentrifuge tube cap. The laser capture microdissection caps can beshipped as-baked (i.e., packaged without post-bake processing) toprotect the laser capture microdissection transfer film and minimizecontamination. The cap, and the configuration in which it is shipped,provides the additional advantages of quick and easy utilization. Thus,it is rendered possible to simultaneously satisfy the requirements ofspeed, accuracy, imaging and resistance to contamination, which, in thecase of the prior art, are mutually contradicting and cannot besimultaneously satisfied.

One aspect of the invention is an apparatus and method of imaging asample with a microscope comprising the step of locating a scatteringmedia within a beam path defined by the microscope and imaging saidsample through the scattering media. The scattering media is opticallycoupled to a laser capture microdissection transfer film. In othervariations, the scattering media is located in or attached to the lasercapture microdissection transfer film or transfer film carrier.

These, and other, aspects of the present invention will be betterappreciated and understood when considered in conjunction with thefollowing description and the accompanying drawings. It should beunderstood, however, that the following description, while indicatingpreferred embodiments of the present invention and numerous specificdetails thereof, is given by way of illustration and not of limitation.Many changes and modifications may be made within the scope of thepresent invention without departing from the spirit thereof, and theinvention includes all such modifications.

BRIEF DESCRIPTION OF THE SEVERAL VIEWS OF THE DRAWINGS

A clear conception of the advantages and features constituting thepresent invention, and of the components and operation of model systemsprovided with the present invention, will become more readily apparentby referring to the exemplary, and therefore nonlimiting, embodimentsillustrated in the drawings accompanying and forming a part of thisspecification, wherein like reference numerals (if they occur in morethan one view) designate the same elements. Consequently, the claims areto be given the broadest interpretation that is consistent with thespecification and the drawings. It should be noted that the featuresillustrated in the drawings are not necessarily drawn to scale.

FIGS. 1A-1C illustrate three views of a laser capture microdissection(LCM) sample plate, representing an embodiment of the present invention;

FIGS. 2A-2C illustrate three views of the sample plate shown in FIGS:1A-1C after coating with a release agent, representing an embodiment ofthe present invention;

FIGS. 3A-3D illustrate four views of a sample carrier, representing anembodiment of the present invention;

FIGS. 4A-4D illustrate four views of the sample carrier illustrated inFIGS. 3A-3D after an LCM film is added, representing an embodiment ofthe present invention;

FIGS. 5A-5C illustrate three views of an assembly that includes four ofthe sample carriers depicted in FIGS. 4A-4D and one of the platesdepicted in FIGS. 2A-2C, representing an embodiment of the presentinvention;

FIGS. 6A-6C illustrate three views of a completed assembly after vacuumhot cast molding, representing an embodiment of the present invention;

FIGS. 7A-7B illustrate two sequential views of a laser capturemicrodissection film with molded features, representing an embodiment ofthe present invention;

FIG. 8 illustrates a bottom view of a laser capture microdissection filmwith molded features, representing an embodiment of the presentinvention;

FIG. 9 illustrates a side view of a laser capture microdissectionapparatus, representing an embodiment of the invention;

FIG. 10 illustrates a side view of a microcentrifuge tube cap with anegative draft, representing an embodiment of the invention; and

FIGS. 11A-11D illustrates a several views of a biological reactionvessel; representing an embodiment of the invention.

DETAILED DESCRIPTION OF THE INVENTION

The present invention and the various features and advantageous detailsthereof are explained more fully with reference to the nonlimitingembodiments that are illustrated in the accompanying drawings anddetailed in the following description. Descriptions of well knowncomponents and processing techniques are omitted so as not tounnecessarily obscure the present invention in detail.

The entire contents of U.S. Ser. No. 60/unknown, filed Feb. 7, 1997(Docket No. ARCT-002); U.S. Ser. No. 08/797,026, filed Feb. 7, 1997;U.S. Ser. No. 08/800,882, filed Feb. 14, 1997; U.S. Ser. No. 60/060,731,filed Oct. 1, 1997; and U.S. Ser. No. 60/060,732, filed Oct. 1, 1997 arehereby expressly incorporated by reference into the present applicationas if fully set forth herein.

Turning to FIGS. 1A-1C, a plate 100 is depicted. Plate 100 can befabricated from metal, glass, ceramic, or any other material suitablefor the subsequent processing steps described below. In a preferredembodiment, plate 100 is a glass microscope slide. It is important thatthe top surface 101 of plate 100 be flat. Although the depictedembodiment shows a bare microscope slide, the plate can be coated, orotherwise surface treated, in a preliminary processing step.

Turning now to FIGS. 2A-2C, the plate 100 is depicted with a releaseagent 210. The release agent 210 is applied to the top surface 101. Itwill be noted that the top surface 101 is obscured by the release agent210 in FIGS. 2A-2B but is clearly visible as an interface in FIG. 2C.

The release agent can be any suitable nonadhesive material such as, forexample, silicones, or TEFLON (i.e., polytetrafluoroethylene).Advantageously, the release coating can be a surfactant that increasesthe contact angle of liquids with which it comes in contact. It isimportant that the release agent 210 maintain and extend the flatnessprovided initially by the top surface 101. In a preferred embodiment,the release agent 210 can include a silicone containing surfactant agentsuch as, for example, RAIN-X.

Turning now to FIGS. 3A-3D, a sample carrier 300 is depicted. The samplecarrier 300 has an upper portion 310 and a lower portion 320. The upperportion 310 includes a top surface 315 and an outer perimeter 317, and ashoulder 319. The lower portion 320 includes a flare 322, an innerperimeter 324, a taper 326 and a substrate surface 328.

The sample carrier 300 can be a polymeric cap that is of transparentoptical quality. For example, the cap could be fabricated frompolycarbonate, or other suitable optically transparent plastic. However,the cap does not need to be optically transparent provided theabsorption characteristics of the polymer from which it is made arecompatible with suitable transmission of the laser energy to the capturefilm.

Turning now to FIGS. 4A-4D, a laser capture microdissection (LCM)transfer film 400 is shown being applied to the sample carrier 300. Itwill be appreciated that the LCM transfer film 400 is depicted out ofscale for the sake of clarity. The laser capture microdissectiontransfer film 400 can be applied to the bottom of a circular cap bypunching a circular section from a free-standing sheet of ethylene vinylacetate. Alternatively, the LCM transfer film 400 can be molded to thebottom of the cap. The LCM transfer film 400 can be deposited on the capusing a process such as spin coating, dipping, or spraying. In anyevent, manufacture of the consumable should be carried out in a sterileenvironment.

It is advantageous that the LCM transfer film 400 be thin. For example,a 50 micron thick film is preferable to a 100 micron thick film.However, the film can advantageously be fabricated in thicknesses ofapproximately 500, 400, 300, 200, 100, 50 microns, or less.

Turning to FIGS. 5A-5C, a plurality of combined sample carriers 300together with their corresponding LCM transfer films 400 are depictedbeing lowered toward the release agent 210 that is coated on top of theplate 100. The LCM transfer films 400 can be an ethylene vinyl acetate(EVA) polymeric material. It will appreciated that FIG. 5A depicts theassembly process at an earlier point in time compared to FIG. 5C whereinthe gap between the LCM transfer film 400 and the release agent 210 isalmost closed.

Turning now to FIGS. 6A-6C, the assembly of four sample carriers 300 onplate 100 is depicted during the process step of vacuum hot baking. Theprocess of vacuum hot baking causes the EVA to soften, melt and flowthereby conforming to the substantially planar surface presented by therelease agent 210. In this way, the flatness possessed by the plate 100is transferred to the LCM transfer film 400. This also eliminatestrapped air.

The hot vacuum baking of the film can take place in moderate vacuum. Ina preferred embodiment, the hot cast molding takes place at one torr and95 degrees C. for approximately one hour.

In an alternative embodiment, instead of attaching the LCM film to thebase of the cap prior to its placement on top of the release agentcoated plate, the LCM film can be coated on top of the release agent asa film layer. A sample carrier can then be placed on top of the LCMfilm. An assembly of one, or more, such combinations can then besubjected to hot vacuum melt casting to planarize at least that portionof the LCM film that is located at the interface between the samplecarrier and the release agent. In this way, when the sample carrier isremoved from the plate, a portion of the planarized LCM film thatcorresponds with the bottom surface of the sample carrier will be brokenaway from the assembly together with the cap that is being removed.Those portions of the LCM film that are not adjacent the bottom of thecap being removed will remain on the plate. In a preferred embodiment,when the sample carrier is pulled away from the plate, a twisting motionis applied to the sample carrier either before and/or during linearseparation of the two prime components so as to exert a sheer force bothwithin the LCM film and between the LCM film and the release layer.

The release coating can be a silicone. Alternatively, the releasecoating can be a polytetrafluoroethylene.

Throughout this specification, the more descriptive phrase “transferfilm carrier” can be substituted for the phrase “sample carrier.” Ingeneral, the transfer film carrier carries the transfer film. Only thatportion of the sample that is transferred to the transfer film iscarried by the carrier.

The ethylene vinyl acetate can be selected from among the availablematerials based on the following criteria. The ethylene vinyl acetateshould have a high melt index. A high melt index is indicated by lowviscosity and low molecular weight.

It is important that the ethylene vinyl acetate, or other material beingused for the LCM transfer film, have a modest tack. Thus, the transferfilm is somewhat sticky but will not bind to everything with which itcomes in contact.

The caps can be made from clear plexiglass G (i.e., polymethylmethacrylate). By treating the glass slide with a surfactant before thecaps are vacuum hot cast in place, the completed caps can be popped offthe glass slide just before they are needed for acquisition of samplematerial.

In a preferred embodiment, the cap is sized to fit in a standardmicrocentrifuge tube. The LCM transfer film can be attached to the capusing glue, or by welding the thermoplastic, or by some other mechanicalmeans, holding the film in place.

The side walls of the cap can have a negative draft. This negative draftcan be machined into the tooling with which the caps are made.

After capturing the tissue to be analyzed on the bottom of the cap, thecap is placed on the microcentrifuge tube containing proteinase (i.e.,protease, e.g., Trypsin) solution and the tube is inverted. The tissueis then dissolved and the DNA is free to enter the solution. Thesolution is then pipetted out of the tube and into the PCR mixture.

While not being bound by theory, it is believed that the EVA filmexpands both up and down when it is exposed to the energy from thelaser. As an approximation, it is believed that the EVA film expandsapproximately 12-15% downward and upward when it is exposed to the LCMcharge from the laser. The upward expansion is restricted by the plasticcap. The thickness of the LCM transfer film should be held to within20%, preferably 5%. The bottom exposed surface of the LCM transfer filmcan be termed a capture surface. The flatness of the LCM transfer filmshould be held to within approximately five microns, preferablyapproximately one micron. The flatness of the film can readilycharacterized based on the number of fringes multiplied by λ/2. Theflatness of the LCM transfer film should preferably be held to withintwo waves which is approximately equal to ¼ micron per fringe, given a λof 540 nm.

The dye in the ethylene vinyl acetate is what absorbs the laser energy.The ethylene vinyl acetate transforms to a liquid phase, infuses intothe cell structure of interest and then hardens.

The particular manufacturing process used for fabricating the assemblyshould be inexpensive and reproducible. Conveniently, the fabrication ofthe present invention can be carried out by using any coating and bakingmethod. It is preferred that the process be conducted in acontaminant-free environment. For the manufacturing operation, it ismoreover an advantage to employ an automated method.

However, the particular manufacturing process used for fabricating theassembly is not essential to the present invention as long as itprovides the described assembly. Normally those who make or use theinvention will select the manufacturing process based upon tooling andenergy requirements, the expected application requirements of the finalproduct, and the demands of the overall manufacturing process.

The particular material used for the cap should be biologically andchemically inert. Conveniently, the cap of the present invention can bemade of any material with a melting point higher than that of EVA. It ispreferred that the material be inexpensive. For the manufacturingoperation, it is moreover an advantage to employ a transparentthermoplastic material that can be injection molded or machined. Forexample, the cap can include polymethyl methacrylate. By properselection of the polymeric materials, the cap can be solid. There is noneed for a through-hole through the center axis of the cap.

However, the particular material selected for the cap is not essentialto the present invention, as long as it provides the described function.Normally, those who make or use the invention will select the bestcommercially available material based upon the economics of cost andavailability, the expected application requirements of the finalproduct, and the demands of the overall manufacturing process.

The LCM transfer film can be any suitable thermoplastic. For example,the LCM transfer film can include one or more of EVAs; polyurethanes(PU); polyvinyl acetates; ethylene-methyl acrylate (EMAC); polycarbonate(PC); ethylene-vinyl alcohol copolymers (EVOH); polypropylene (PP); andexpandable or general purpose polystyrene (PS). ELVAX 410, 200 and 205are suitable resins of EVA that are commercially available from DuPontwherein the operative variant is the amount of vinyl.

The LCM transfer film can include an absorptive substance. Theabsorptive substance can include an absorptive dye. This dye can beeither a broad band absorptive dye or a frequency specific absorptivedye. For example, the absorptive dyes can include one or more of tin(IV)2,3-naphthalocyanine dichloride; silicon(IV) 2,3-naphthalocyaninedihydroxide; silicon (IV) 2,3-naphthalocyanine dioctyloxide; and vanadyl2,11,20,29-tetra-tert-butyl-2,3-naphthalocyanine. Also, the absorptivesubstance can include a plurality of Fullerines (i.e., Bucky Balls,e.g., C60).

The LCM transfer film can also include a scattering media. Since the LCMtransfer film is very close to the sample, the scattering media reducesshadows, thereby improving the process of imaging. The scattering mediacan include a diffusing material. For example, the LCM transfer film canbe loaded with a small particulate material that scatters theillumination light so as to minimize shadows and improve imaging withoutdetrimentally affecting the LCM beam. Alternatively, the transfer filmcan include a dichromatic gelatin (DCG) to perform the same functions.The DCG can be exposed and developed to provide specific diffuserproperties within the transfer film such as shaping.

There are a variety of techniques for building a noncontact LCM transferfilm and/or carrier. The purpose of the noncontact LCM approach is toprovide a method for the elimination of problems associated withnonspecific binding of tissue to an LCM film. In more detail, if asample slide has areas with loosely attached cells, these portions ofthe sample can be lifted mistakenly from the slide due to nonspecificattachment to the LCM film. That is, these areas stick to the film eventhough they were not illuminated by the laser. If these portions aretransferred to the reagent vessel they will be digested by the reagentsand appear as contaminants in the sample. It is important to prevent theloosely bound tissue areas from contacting the film.

One method for preventing the contact of the film to areas of tissuethat might nonspecifically transfer is to offset (distance) the film afew microns from the tissue sample. In the area illuminated by thelaser, the film expands roughly 10% of its thickness (about 5 to 10microns based on a typical thickness of 50 to 100 microns) and contactsthe tissue, thereby allowing transfer in the illuminated region. Outsidethis region, the film and tissue never come in contact because the filmis spaced away from the tissue. The film, however, must not be spacedtoo far from the tissue (greater than a few microns) since the filmneeds to contact the tissue after it expands due to the laserillumination.

One technique to make a noncontact LCM transfer film that “stands-off” afew microns is to create a series of pedestals that are a few micronshigh so as to provide a series of standoffs for the cap to rest on.These pedestals can be created by exposing edges of the transfer film tothe focused laser beam. The laser beam distorts the normally flat filmin the focal region raising the surface in this region. By placing thesepedestals at the vertices of an equilateral triangle with points locatedat the rim of the transfer film carrier a good three-point mount isprovided. The height of these pedestals can be adjusted by changing thepower and pulse length of the focused laser beam. The diameter can beadjusted by changing the diameter of the laser beam. The exposure levelsare similar to the levels used for tissue transfer: approximately 10-90mW for approximately 10-90 milliseconds. (To create the pedestals it mayhelp to expose the film when it is in contact with a glass slide.) Thereagent vial can be constructed so that it has an internal rim thatcontacts the pedestals, sealing them from the reagent, therebypreventing tissue that might be on the pedestals from contaminating thesample.

Turning now to FIGS. 7A-7B, an LCM film 700 can be provided withfeatures 710. The features 710 can include a raised portion 720(pedestal) and a protruding feature 730 (e.g., rim). The features 710can be molded (e.g., replicated), or otherwise formed (e.g., by laser),in the LCM film 700. Such features give the LCM film 700 a workingsurface that defines a topography.

The purpose of the features 710 is to provide an additional way ofselecting single cells from, a tissue sample using LCM, other than justa very small laser spot size. The features 710 that are fabricated intothe LCM transfer film can be roughly the size of a desired cell 740. Thefeatures 710 can extend out from the film surface for a distance ofseveral microns.

The film 700 itself can be offset from the cells a distance of fromapproximately 5 to approximately 10 microns by the protruding feature730 that runs around the circumference of the cap. To stabilize theplane of the film, it will be appreciated that the protruding featureonly needs to extend along at least three points of a perimeter of thefilm and does not need to be a continuous rim.

The features 710 can be fabricated by hot cast molding the LCM film 700against a mold that has complimentary shapes of the features lasermachined into the mold surface. Such a mold can be made out of apolished metal surface or a glass surface using a Q-switched laserfocused to a diameter of from approximately 5 to approximately 20microns. The features 710 can also be fabricated by molding the filmagainst a mold surface that is micro machined with a diamond stylus. Thetopography is transferred from the mold to the film via replication.

A protuberance (raised portion 720) for acquiring the desired cell 740can include a small raised area of LCM film roughly 5 to 20 microns indiameter. When a laser beam 750 heats this portion of the film, theraised portion 720 will contact the tissue first and the laser power canbe adjusted so that the surrounding adjacent film regions do not contactthe tissue. Thus, the raised portion 720 provides spatial discriminationin addition to the spatial discrimination provided by the position, sizeand mode of the laser beam. An advantage of the features 710 is that alarger laser beam could be used and a researcher or laboratorytechnician could still achieve single cell lift-off. The raised portionof the film (raised portion 720) will be heated to a higher temperaturethan the surrounding flat film area. The protruding feature 730 (i.e.,the rim) will not be heated. This would also increase the likelihoodthat a cell in the region of the feature would be captured exclusively.Of course, it is advantageous that raised portion 720 not protrude asfar as protruding feature 730.

Referring now to FIG. 8, multiple pedestals 800 could be molded into anLCM film 810 to allow multiple single cell lift off regions. The LCMfilm 810 could again include a rim 820. Multiple cells could then beanalyzed in a single microcentrifuge tube.

The structural feature (i.e., spacer) that holds the film away from thesample can be hot vacuum baked into the transfer film. According to thisprocess, a negative of the structural feature can be formed in a plate.The structural feature is then replicated (as a positive) in the filmwhen it is heated and flows into the void defined by the negative of thefeature. Alternatively, the structural feature can be formed in thetransfer film with the use of a laser, or even with micro-machiningequipment.

The structural feature, or spacer, can be integrally formed in the lasercapture microdissection transfer film. The structural feature provides aseparation between the transfer film and the sample. This separationholds the film away from the sample, thereby enabling noncontact lasercapture microdissection.

The transfer film can be connected to the substrate surface with arefractive index matching transparent fluid or glue. Alternatively, thetransfer film can be coupled to the substrate surface by punching boththe sample carrier and the transfer film from stock materialsimultaneously. It is even possible to couple the film to the carrierwith double sided tape.

The laser capture microdissection transfer film includes a substantiallyplanarized low land area. This low land area can be provided withstructural features that protrude so as to define a laser capturemicrodissection acquisition zone. These protrusions can be termedpedestals. The low land can also be provided with structural featuresthat hold most of the film away from the sample. In order to support theplane of the film, it is preferable to have at least three suchsupporting features. If these supporting features run around most, orall, of a perimeter of a transfer film, they can be termed a rim.

Whatever contacts the tissue needs to be equidistant from the tissue sothat the dosimetry is constant across the transfer film. In this way, aknown distance between the tissue and the transfer film can beestablished. In many cases such a known distance will be fixed acrosssubstantial portions of the transfer film surface. However, it issufficient that the distance be known and does not need to be fixed. Thedistance needs to be known for the purpose of adjusting laser power soas to achieve tissue transfer.

When the transfer film is exposed to the electromagnetic energy, itexpands (both up and down) against the substrate surface and contactsthe tissue, thereby injecting itself into the sample. In the case wherethere is a space between the transfer top surface of the sample,(noncontact laser capture microdissection) the expanding film will beprojected through that space before it contacts the top surface of thesample at the beginning of the injection phase.

Referring now to FIG. 9, a scatter illuminator design for an LCM deviceis illustrated. The purpose of the scatter illuminator design is toprovide a more appropriate illuminator for an LCM microscope thatgenerates a more even illumination to prevent shadows from obscuringinternal cell structure.

A laser capture microdissection apparatus includes a top portion 910 anda bottom portion 920. The top portion 910 includes an upper surface towhich a scattering media 930 can be coupled. The bottom portion 920includes a substrate surface to which a scattering media 940 can becoupled. Either, or both, of the scattering media 930 and 940 can beused. The scattering media can be incorporated into the transfer filmcarrier and/or the LCM transfer film.

Using a standard inverted microscope light source and placing ascattering media (e.g., a piece of paper) near the tissue to scatter thelight results in dramatically improved illumination of the sample andmuch better visualization. A scattering media of this type eliminatesthe need for refractive index matching of the sample. Such a scatteringmedia can allow visualization of the cell nucleus and other subcellularstructures that would normally be obscured by normal illuminationtechniques.

The scattering media can be a diffuser material. A diffuser materialthat is suitable for use as the scattering media is milk glass which isa very dense, fine diffuser available from Edmund Scientific as Part No.P43,717. Standard laser printer/photocopier paper can even be used asthe scattering media. Other types of transparent scattering media can beused, such as, for example, frosted glass, a lenticular sheet, a volumediffuser, and/or a surface diffuser. In any event, the scattering mediashould be a material that aggressively scatters the illumination light.A single sheet of typical ground glass is generally inadequate and needsto be combined in multiple layers as a serial stack of three or foursheets of ground glass to diffuse the illumination light sufficiently.

The scattering media can be directly or indirectly connected to thetransfer film carrier and/or the LCM transfer film. Alternatively, thescattering media can be formed on a surface of, or the interior of, thetransfer film carrier and/or the LCM transfer film. The scattering mediacan be fabricated so as to shape the LCM beam and/or the illuminationbeam. The scattering media needs to be within a few millimeters of thesample to be effective. A few millimeters means less than onecentimeter, preferably less than five millimeters.

Referring now to FIG. 10, a laser capture microdissection apparatus 1000is illustrated. The apparatus 1000 includes a top portion 1010 and abottom portion 1020. The bottom portion 1020 includes a negative draft1030. The negative draft 1030 is preferably approximately 5°. The bottomportion 1020 also includes a chamfer 1040. The chamfer 1040 ispreferably approximately 20°. The bottom portion 1020 also includes agirdle 1050. The width of the girdle 1050 for line contact with theinterior of an analysis vessel is preferably approximately 0.01 inches.Caps with a negative draft can be fabricated with a break-apart plasticinjection molding die. Alternatively, negative draft caps can befabricated by interpolation with computer numeric control cutting toolmachinery.

Turning now to FIGS. 11A-11D, a laser capture microdissection (LCM)biological reaction vessel 1100 including an analysis vessel 1110 withan internal ridge and a cap 1120 with a transfer film 1130. The transferfilm 1130 can include EVA and can have a stand-off rim 1150. Stand-offrim 1150 can be a 10-20 micron ridge providing a noncontact region inthe center of the transfer film 1130. The cap 1120 is an integralportion of the biological reaction vessel 1100. The analysis vessel 1110is formed to include an internal ridge. The internal ridge slopes backtoward an opening in the analysis vessel 1110 so as to make a tight sealwith the cap 1120, even if the stand-off rim is not present. The purposeof combining the internal ridge 1140 with the stand-off rim 1150 in asingle embodiment is to provide an LCM analysis vessel and film carrierthat have features to facilitate a noncontact method for positioning thetransfer film over the tissue sample. The LCM non-contact method reducesthe probability that areas of tissue outside the focal adhesion regionwill be transferred. However, if the stand-off rim 1150 comes in contactwith the reaction, this advantage will be lost. The analysis vessel 1110with this internal sealing feature allows the transfer film 1130, withstand-off rim 1150, to contact reaction fluid in the analysis vessel1110.

The biological reaction vessel 1100 includes the cap 1120 (lid) that canbe removably coupled to the analysis vessel 1110. The transfer film 1130is attached to the clear plastic cap 1120. The transfer film 1130 can behot cast molded to include the stand-off rim 1150 that is 10 micronsthicker than the central region of the cap 1120. The stand-off rim 1150can be termed an annular rim. The transfer film 1130 expands in theregion of the focused laser beam and is able to bridge the 10 microngap, thereby contacting the tissue and allowing transfer of a portion ofthe tissue to the film. This stand-off rim 1150 can be termed a standoffregion and acts as a spacer elevating the central region of the transferfilm 1130 above the tissue and preventing the transfer film 1130 fromcontacting the tissue in this central region, until the LCM laseractivates the transfer film 1130. This stand-off region feature can bemolded into the transfer film 1130 by pressing the transfer film 1130onto a heated plate that contains an inverse image of this step (spacer)feature. This method replicates the feature. Such a mold could beconstructed using a polished metal plate and standard chemical etchingtechniques. It could also be manufactured using glass or siliconsubstrates and chemical etching. Alternatively, a diamond lathe could beused to machine this feature onto a suitable metal substrate (e.g.,copper, aluminum, steel, etc.).

The cap 1120 that seals the liquid reagent analysis vessel 1110 can bemade out of inert plastic such as polypropylene or polyethylene. Theanalysis vessel 1110 has the internal ridge 1140 (step) that is designedto mate with and cover the annular rim of the cap 1120 providing a tightseal at this point. This seal prevents liquids in the analysis vessel1110 from contacting the bottom surface of the rim of the cap. Thisdesign eliminates nonspecific tissue transfer since the stand-off rim1150 is the only area of the cap 1120 that contacts the tissue (otherthan the desired transfer regions illuminated by the laser) and thedigestion reagents in the analysis vessel 1110 never contact this region(stand-off rim 1150). The internal ridge 1140 feature in the analysisvessel can be designed with a slight angle so as to partially cut intothe transfer film 1130 providing a very tight seal similar to vacuumflange sealing techniques. A slight bulge or indentation can be moldedinto the barrel of the cap 1120 or into the top portion of the analysisvessel 1110 so as to provide a downward directed force and a positiveseal between the cap 1120 and the analysis vessel 1110.

EXAMPLE

A specific embodiment of the present invention will now be furtherdescribed by the following, nonlimiting example which will serve toillustrate in some detail various features of significance. The exampleis intended merely to facilitate an understanding of ways in which thepresent invention may be practiced and to further enable those of skillin the art to practice the present invention. Accordingly, the exampleshould not be construed as limiting the scope of the present invention.

In an exemplary embodiment of the invention, a glass microscope slide isfirst cleaned. Then the glass microscope slide is spray coated with athin layer of a commercially available silicone release agent, in thisexample a silicone containing surfactant that is readily commerciallyavailable (i.e., RAINEX). Meanwhile, a supply of sample carriers in theform of microcentrifuge tube caps are molded from plexiglass G.Cylindrical chips of LCM film punched from a sheet of ethylene vinylacetate (EVA) are then attached to the bottom surface of the caps,optionally with an epoxy adhesive. The resultant cap subassemblies arethen placed on top of the release agent coated glass subassembly for hotvacuum baking. The hot vacuum baking is carried out at a pressure ofapproximately one torr or less at a temperature of 95° C. forapproximately one hour. This planarizes the transfer film. The bakedassembly is then allowed to cool to room temperature. The resultingassembly can include a plano-concave void located between each of thecaps and the underlying plate. In this way only the perimeter of thebottom of the caps is in contact with the glass plate. This provides twosignificant advantages. First, the working surface of the LCM film isspaced apart from the glass slide in a vacuum and remains free ofsurface damage and contaminants. Second, the removal of each cap fromthe glass slide is facilitated by the fact that only a fraction of thesurface area of the bottom of the cap is attached to the release layerthat has been coated on the glass slide. Therefore, removal of the capfrom the slide requires much less force than if the entire lower surfaceof the cap were in contact with the release layer.

It can be appreciated that by both making and shipping the cap on thesame glass slide, the number of processing and packaging steps isreduced while reproducibility and cleanliness are improved.

The completed consumable products can be sterilized (e.g., with beta orgamma radiation). Finally, the completed consumable products should besubjected to a rigorous quality assurance inspection.

There are a number of advantages to leaving the caps on the slide untilthey are about to be used. These advantages include protection of theoptically flat surface. For example, leaving the caps on the slidereduces hydroxyl contamination of the transfer film. These advantagesalso include the prevention of particulate matter from settling on thesurface.

Practical Applications of the Invention

A practical application of the present invention that has value withinthe technological arts is the collection of a large database of geneexpression patterns of both healthy and diseased tissue at differentstages of diseases. This database will be used to more fully understandthat pathogenesis of cancer and infectious diseases. The presentinvention will enable a scientist to identify gene patterns andincorporate this information into effective diagnostics for disease. Thepresent invention will allow medical doctors to compare actual patienttissue samples with archived data from patient samples at differentdisease stages, thereby allowing them to prescribe more effective stagetherapies, eliminate unnecessary procedures, and reduce patientsuffering. Other research areas where the present invention will finduse are drug discovery, developmental biology, forensics, botany, andthe study of infectious diseases such a drug-resistant tuberculosis.There are virtually innumerable uses for the present invention, all ofwhich need not be detailed here.

Advantages of the Invention

Laser capture microdissection, representing an embodiment of theinvention can be cost effective and advantageous for at least thefollowing reasons. The present invention will replace current methodswith better technology that allows for more accurate and reproducibleresults. The present invention can be used to provide a low costinjection molded polymer disposable that integrates a laser capturemicrodissection film into the interior surface of an analysis containersuch as a microcentrifuge tube.

All the disclosed embodiments of the invention described herein can berealized and practiced without undue experimentation. Although the bestmode of carrying out the invention contemplated by the inventors isdisclosed above, practice of the present invention is not limitedthereto. It will be manifest that various additions, modifications andrearrangements of the features of the present invention may be madewithout deviating from the spirit and scope of the underlying inventiveconcept. Accordingly, it will be appreciated by those skilled in the artthat the invention may be practiced otherwise than as specificallydescribed herein.

For example, the individual components need not be formed in thedisclosed shapes, or assembled in the disclosed configuration, but couldbe provided in virtually any shape, and assembled in virtually anyconfiguration. Further, the individual components need not be fabricatedfrom the disclosed materials, but could be fabricated from virtually anysuitable materials. Further, although the caps and cap assembliesdisclosed herein are described as a physically separate module, it willbe manifest that the caps and cap assemblies may be integrated intoother apparatus with which they are associated. Furthermore, all thedisclosed elements and features of each disclosed embodiment can becombined with, or substituted for, the disclosed elements and featuresof every other disclosed embodiment except where such elements orfeatures are mutually exclusive.

It is intended that the appended claims cover all such additions,modifications and rearrangements. The claims are not to be construed asincluding means-plus-function limitations, unless such limitations areexplicitly recited using the term “means” in the claims. Expedientembodiments of the present invention are differentiated by the appendedsubclaims.

1. A method of imaging a sample with a microscope, comprising: providingsaid microscope; locating a scattering media within a beam path definedby said microscope; and imaging said sample through said scatteringmedia with said microscope.
 2. The method of imaging a sample with amicroscope according to claim 1, wherein said scattering media isoptically coupled to a laser capture microdissection transfer film. 3.The method of imaging a sample with a microscope according to claim 1,further including the step of providing a transfer film carrier; andwherein said scattering media is attached to an upper surface of saidtransfer film carrier.
 4. The method of imaging a sample with amicroscope according to claim 1, further including the step of providinga transfer film carrier; and wherein said scattering media is attachedto a substrate surface of said transfer film carrier.
 5. The method ofimaging a sample with a microscope according to claim 1, furtherincluding the step of providing a transfer film carrier; and whereinsaid scattering media is attached to said transfer film carrier.
 6. Themethod of imaging a sample with a microscope according to claim 1,further including the step of providing a transfer film carrier; andwherein said scattering media is located in said transfer film carrier.7. The method of imaging a sample with a microscope according to claim1, further including the steps of: providing a transfer film carrier;providing a laser capture microdissection transfer film attached to saidtransfer film carrier; and wherein said scattering media is located insaid laser capture microdissection transfer film.
 8. The method ofimaging a sample with a microscope according to claim 1, furtherincluding the steps of: providing a transfer film carrier; providing alaser capture microdissection transfer film attached to said transferfilm carrier; and wherein said scattering media is located on a surfaceof said laser capture microdissection transfer film.
 9. The method ofimaging a sample with a microscope according to claim 1, furtherincluding the step of providing a laser capture microdissectioninstrument.
 10. A method of imaging a sample with a microscope,comprising the step of positioning said scattering media within a fewmillimeters of a sample.
 11. A microscope, comprising: a scatteringmedia located within a beam path defined by said microscope and within afew millimeters of a sample.
 12. The microscope of claim 11, furthercomprising a laser capture microdissection film optically coupled tosaid scattering media.
 13. The microscope of claim 11 furthercomprising: a transfer film carrier; said scattering media attached toan upper surface of said transfer film carrier.
 14. The microscope ofclaim 11 further comprising: a transfer film carrier; said scatteringmedia attached to a substrate surface of said transfer film carrier. 15.The microscope of claim 11 further comprising: a transfer film carrier;said scattering media attached to said transfer film carrier.
 16. Themicroscope of claim 11 further comprising: a transfer film carrier; saidscattering media located in said transfer film carrier.
 17. Themicroscope of claim 11 further comprising: a transfer film carrier; alaser capture microdissection transfer film attached to said transferfilm carrier; said scattering media located in said laser capturemicrodissection transfer film.
 18. The microscope of claim 11 furthercomprising: a transfer film carrier; a laser capture microdissectiontransfer film attached to said transfer film carrier; said scatteringmedia located on a surface of said laser capture microdissectiontransfer film.
 19. The microscope of claim 11 further comprising a lasercapture microdissection instrument.
 20. The microscope of claim 11wherein said scattering media is located within a few millimeters of asample.